"Antibody-derived single chain fragment variable (scFv) and its use for the identification of the extracellular portion of CD79B for therapeutic and diagnostic purposes"

Inventors' name: Brunangelo Falini, Vincenzo Maria Perriello kkk

FIELD OF THE INVENTION

1.The present invention relates to the field of antibodies and their application for diagnostic and immunotherapeutic purposes in onco-hematology.

2.In particular, the present invention relates to the development of a monoclonal antibody, of the G type immunoglobulin group, capable to recognize and bind the extracellular portion of the human CD79B protein (Figure 1) and to its medical use in the immunotherapy field of lymphomas expressing CD79B through the single chain fragment variable (scFv) region of the antibody.

STATE OF THE ART /BACKGROUND

3.The availability to develop monoclonal antibodies against antigens expressed by neoplastic cells has revolutionized the diagnosis and treatment of oncological diseases.

4.Furthermore, in recent times, the increase in knowledge in the field of immuno-oncology and genetic engineering has made it possible to exploit the characteristic specificity of monoclonal antibodies to produce new models of immunotherapy such as bi-/tri-specific antibodies, nanobodies and CAR-T cells (chimeric antigen receptor T-cells). These innovative approaches of immunotherapy are based upon the combination between the specific recognition of a specific tumor protein, typical of antibody-mediated immune response, and the cytotoxicity induced by lymphocyte activation, characteristic of cell-mediated immune response. The combination of these two potentialities allows cytotoxic T lymphocytes to eliminate tumor cells that express a certain extracellular antigen, in a selective and specific way. The recognition of tumor antigens in these new immunotherapy approaches does not occur in a physiological way, through the interaction between the T-cell receptor (TCR) and the major histocompatibility complex, but is mediated by a sequence of artificial amino acids, derived from the variable light and heavy chains regions (named complementary determining regions, CDRs) of a monoclonal antibody. This sequence is called the single-chain fragment variable (scFv) region and it is obtained by conjugating together the CDRs of the genetic sequence of a specific monoclonal antibody. The advantage of scFv-mediated antigen-specific recognition is the possibility to generate an immune response against any molecule expressed on tumor cells surface, even if not presented by the major histocompatibility complex. The opportunity to eliminate these cells, "invisible" to the immune system for the major histocompatibility complex downregulation, constitutes an important therapeutic option in neoplastic patients relapsed or refractory to chemo and/or radiotherapy . . An example of how these new forms of immunotherapy are particularly effective is represented by CAR-T cells, in which the patients' T lymphocytes are genetically modified to express the chimeric protein CAR, formed by the fusion between the scFv of a monoclonal antibody and the activation domain of T cells (CD3z). . The recognition of a specific antigen expressed by the tumor cell by the chimeric CAR protein, determines the activation of the transduced lymphocyte with the consequent release of granules and cytokines that lead to the selective destruction of the target cell, even in the absence of the antigen presentation by the major histocompatibility complex. Several clinical studies have shown the considerable success of CAR-T cell therapy in the onco-hematological field, especially through the use of anti-CD19 CAR T cells for the treatment of some lymphoproliferative diseases such as acute lymphoblastic leukemia and diffuse large B cell lymphomas.

12. CD19 antigen is considered an almost ideal target for novel immunotherapy approaches using CAR-T cells as it is expressed in most neoplastic cells, while it is not expressed on normal cells except in mature B lymphocytes. However, a limitation of anti-CD19 CAR-T cell therapy is the possible phenomenon of "antigen escape", due to the ability of the tumor cell not to express or express the CD19 antigen on the cell surface in a different way by alternative splicing.

13. This phenomenon allows CD19 negative tumor cells to escape recognition and cytotoxic action of anti-CD19 CAR-T cells, and, therefore, to proliferate and cause disease recurrence. It is, therefore, necessary to identify new target antigens, important for tumor cell proliferation and survival thus reducing the possibility of an "antigen escape" mechanism, allowing a more effective and lasting response after CAR-T cell treatment.

14. Because of the small number of alternative tumor antigens to CD19 for the treatment of B lymphoproliferative diseases with CAR-T cells, the production of new monoclonal antibodies is essential to generate the scFvs necessary for the development of novel CAR-T cells.

15. For this purpose, these new monoclonal antibodies should have the following requirements:

- Recognize a target antigen expressed in the majority of B lymphoproliferative neoplasms cells;

- Recognize an epitope of the extracellular portion of the target antigen in a selective and specific way;

- Recognize a target antigen that possibly has an important role for the tumor cell, which therefore is stably and homogeneously expressed on the cell surface, even in disease relapses;

- Recognize all the isoforms of a target antigen, to avoid "antigen escape" phenomena due to alternative splicing mechanisms. THE PROPOSED SOLUTION

16. The present invention relates to the development of a new monoclonal antibody against the extracellular portion of the human CD79B antigen, for its use in the field of immunotherapy through its scFv. CD79B, also known as B29 or B-cell receptor- associated protein (BCR; Figure 1) beta chain, is considered an important target antigen for immunotherapy approaches in B-cell non-Hodgkin's lymphomas (B cell Non-Hodgkin Lymphoma, B-NHL). CD79B is a highly specific tumor antigen, as it is expressed in most B-NHLs, regardless of stage, subtype, cytogenetic and molecular characteristics.

17. Conversely, among normal cell populations, it is expressed only by mature B lymphocytes, thus reducing the risk of "on- target / off-tumor" toxicity.

18. A clinical study evaluating the safety and activity of polatuzumab vedotin, an anti-CD79B antibody conjugated to the vedotin chemotherapy, in combination with chemotherapy and rituximab, showed that the addition of polatuzumab vedotin did not lead to greater toxicity than chemotherapy alone.

19. Another feature that makes CD79B a particularly promising target for B-NHL immunotherapy is its importance in supporting neoplastic proliferation and in giving lymphomatous cells the ability to be resistant to molecular drugs, such as Ibrutinib.

20. Finally, CD79B is stably expressed on the cell membrane of tumor cells, even when patients relapse after therapies with anti-CDl9 CAR-T cells.

21. It is estimated that approximately 20-30% of relapses after anti-CD19 CAR-T cell therapy in B-NHL are caused by loss of expression of the CD19 antigen. The specific mechanisms responsible for the "antigen escape" are generally due to mutations of exons and splice isoforms of the CD19 gene, which lead to the disappearance of the expression of the epitope recognized by the scFv of the anti-CD19.

22. Therefore, it is fundamental to develop new CAR-T cells against tumor antigens which are conserved in the various stages of the disease. Like CD19, expression of the CD20 antigen may also be lost in B-NHLs as a result of immunological pressure exerted by the anti-CD20 monoclonal antibody Rituximab, frequently associated with chemotherapy regimens in the treatment of lymphoproliferative diseases. CD79B antigen is instead constantly present at the immunohistochemical and immunophenotypic evaluation in B-NHL, both at diagnosis and at relapse. This evidence was also confirmed in a case of diffuse large B cell lymphoma, treated with anti-CD19 CAR-T cells at the Hematology Center of Perugia.

23. After an initial response to anti-CD19 CAR-T cell therapy, documented by PET-CT scan, the patient relapsed in the abdominal area, nearby but previously unaffected by the disease. A biopsy of the new lymph node mass confirmed the presence of lymphoid cells of large size but which on immunohistochemistry no longer expressed the classical markers of B lymphomas such as CD19, CD20, and CD79a (present before therapy with CAR-T cells).

24. The only marker remained constantly expressed before and after CAR-T cell therapy it was CD79B. RNA sequencing comparison between samples before CAR-T cell therapy and at relapse, confirmed the disappearance of CD19 expression and the persistence of CD79B, corroborating the importance of CD79B in the choice of the therapeutic target (Figure 2).

25. Therefore, the development of novel anti-CD79B CAR-T cells could represent a valid therapeutic approach for the treatment of B-NHL. It is therefore of considerable importance to have highly related and specific anti-CD79B monoclonal antibodies for therapeutic purposes, such as CAR-T cell therapies.

26. Furthermore, monoclonal antibodies developed against the extracellular portion of CD79B capable of recognizing both isoforms, could be used for diagnostic purposes as part of the immunohistochemical and immunophenotypic evaluation of all Non- Hodgkin B Lymphomas.

27. In particular, adequately recognizing the expression of CD79B on lymphoma cells could be particularly useful in guiding the choice of the best therapy in a targeted manner, reserving or excluding immunotherapies anti-CD79B such as Polatuzumab based on the presence or absence of CD79B.

DETAILED DESCRIPTION OF THE INVENTION Generation and selection of the anti-CD79B monoclonal antibody

28. The 128 4C5 anti-human CD79B monoclonal antibody was generated against al6 kDa his-conjugated recombinant peptide (U5623EH200-1), corresponding to the aminoterminal extracellular portion NH2 of the CD79b protein (Figure 3).

29. The nucleotide sequence of the recombinant peptide U5623EH200-1 was cloned into the plasmid pET-30a, expressed in E. coli, and purified from bacterial lysates by column chromatography on Ni columns.

30. After sterilization, with a 0.22 pm filter, peptide concentration was determined by protein analysis according to Bradford .

31. The purity of the peptide U5623EH200-1 and its molecular weight were determined by SDS-PAGE standard and confirmed in Western blotting (Figure 4) (GenScript USA Inc, Piscataway, NJ, USA) .

32. The amino acid sequence of the recombinant peptide is the following :

ARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDEN PQQLKLEKG RMEESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQR NTLKD .

33. Monoclonal antibodies (mAb) were produced at the Onco- Hematological Research Center (CREO), Perugia, Italy. Shortly, the recombinant peptide of CD79B extracellular portion (172.5 pg) was resuspended in PBS and administered intraperitoneally to Balb/c mice. After the fourth administration, the mice were sacrificed and their spleen used to generate hybridomas.

34. Among all clones screened by immunohistochemistry, the supernatant of a single hybridoma colony (clone 128) showed reactivity against B lymphocytes on human tonsils frozen sections (Figure 5). Clone 128 was later subcloned 4 times to ensure monoclonality (clone 1284C5).

Evaluation of the specificity of the monoclonal antibody anti- CD79B

35. To evaluate the specificity of anti-CD79B 128 4C5 monoclonal antibody, western blotting and flow cytometry analysis were performed on CD79b positive cells, such as B lymphocytes and two cell lines of non-Hodgkin B lymphoma, SUDHL- 16 and RIVA. T lymphocytes and the acute myeloid leukemia KG-1 cell line, were used as CD79B negative controls. Reactivity against CD79B protein in western blotting was confirmed by comparing the designated 1284C5 mAb with an already validated commercial anti-CD79B mAb, as control. A 37-40 kDa band (corresponding to the molecular weight of CD79B) was detected for lysate human B ymphocytes and B-NHL SUDHL-16 and RIVA cell lines. In all negative controls, no expression was observed in T lymphocytes lysates and KG-1 cell lines. A similar expression profile was detected also with the already validated commercial anti-CD79B antibody, confirming the specificity of 1284C5 antibody (Figure 6). The selective recognition of the CD79B extracellular portion was evaluated by flow cytometry on non-permeabilized human NHL cell lines (SUDHL-16 and RIVA), grown in 20% of FBS 1630 RPMI medium. The cells were washed with a 2% PBS-BSA buffer solution, stained firstly with the designated primary antibody 128 4C5 and then with the secondary antibody conjugated with the Alexa- 488 fluorochrome. Non-permeabilized stained cells were analyzed by BD FACS Canto II instrument, showing intense expression of CD79B compared to stained cells with only the secondary antibody. Furthermore, to exclude cross-reactivity between anti-

CD79B 128 4C5 antibody and other cell surface epitopes, two further investigations were carried out a confirmation of mAb specificity. In the first investigation, the CD79B antigen cDNA was transiently transfected in human embryonic 293t cell lines, which do not express CD79B due to their own fibroblastic derivation. 24 hours after cDNA lipotransfection, 293t cells were stained and analyzed for WB and flow cytometry, as previously described. The anti-CD79B 1284C5 monoclonal antibody showed a marked intensity of expression only in 293t cells transfected with CD79B cDNA, while no reactivity was detected in the negative control 293t non-transfected cells (Figure 7). 43. In the second investigation, gene editing by CRISPR-CAS9 was exploited to knockout (KO) CD79b on SUDHL-16 B-NHL cell line. Briefly, sgRNAs chemically modified to recognize the CD79B gene were designed using using Synthego CRISPR Gene KO software and purchased from Synthego. 1.5 pg of Cas9 protein (IDT) and 1 pg of sgRNA were conjugated and incubated for 15 minutes at room temperature. 400,000 SUDHL-16 cells were washed with PBS, resuspended in Neon T buffer with the Cas9 / sgRNA ribonucleoprotein complex and then electroporated (Invitrogen Neon Electroporation System, Thermofisher, USA).

44. After electroporation, cells were incubated at 37 °C until evaluation. Both western blotting analysis and flow cytometry showed that the designated 1284C5 anti-CD79b mAb only recognize the SUDHL-16 wild type cell line.

45. In the CD79B KO modified cell line, on the other hand, the antibody did not show any reactivity both in flow cytometry and western blotting assays (not recognized bands at the height of the CD79B molecular weight) (Figure 8).

Evaluation of the recognition of both isoforms of CD79B

46. Once established specificity and selectivity of the designated 1284C5 anti-CD79B monoclonal antibody, the capacity to recognize both CD79B antigen isoforms was investigated. 293t cells were transfected with both isoform 1 (also called long isoform, 229 AA length and 33-40 kDa molecular weight) or isoform 2 of the antigen (also called short isoform, with a length of 125 AA and a molecular weight of 15-20 kDa).

47. Cell lysates of transfected 293t cells were evaluated by western blotting analysis with the designated 128 4C5 anti-

CD79b monoclonal antibody compared to non-transfected 293t cells, used as a control. A band at the molecular weight both CD79B isoforms was clearly visible in the lysates of 293t cells transfected, while no band was detectable in the non- transfected 293t cells used as control (Figure 9).

Isolation and sequencing of the antibody scFv monoclonal anti-CD79b Due to the characteristics of this designated mAh 1284C5 against the extracellular portion of the CD79B antigen, one of its own possible therapeutic application could be the use of its scFv for the development of anti-CD79B therapeutic antibodies or chimeric antigen receptors (CARs). For the latter purpose, 1284C5 hybridoma cells were used to extract the genetic sequence of the variable domains of the heavy and light chain of the antibody (regions that determine the complementarity, CDR), characteristic for the specific antigen binding. In short, from 1284C5 hybridoma cells, total RNA was extracted following the technical manual of the RNeasy Plus Micro kit (QIAGEN, Cat. No .: 74034). The RNA was then back-transcribed into cDNA using isotype specific anti-sense primers or primers universal following the technical manual of the transcript reverse SMARTScribe (TaKaRa, Cat. No .: 639536). The fragments of heavy chain and light chain antibodies were amplified according to the rapid amplification procedure of the end of GenScript cDNA (RACE). The fragments of the amplified antibodies were cloned separately into a standard cloning vector and performed a PCR for the screening of clones with correctly sized inserts (Figure 10). The monoclonal antibody of the present invention is also suitable for use in the diagnostic field, in consideration of previously illustrated features. In addition to clearly and specifically recognizing the CD79B positive cells in flow cytometry, the designated 1284C5 antibody was also reactive in immunohistochemistry, on paraffinated sections of B-NHL (Figure 11, top panel). This pattern was similar to a commercial anti-CD79B antibody (Figure 11, panel in low), confirming the possibility of the diagnostic use of this monoclonal antibody also in histology as well as in flow cytometry and immunofluorescence.